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ChIP on ChIP. Students: Jaimie Vettichira Araj Sidki Roshni Gandhi Sonia George Diana Barayeva Edward Salib Instructor: Dr. Claude E. Gagna. DNA Microarrays. What are they? Microscopic DNA spots representing single genes

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chip on chip

ChIP on ChIP

Students: Jaimie Vettichira

Araj Sidki

Roshni Gandhi

Sonia George

Diana Barayeva

Edward Salib

Instructor: Dr. Claude E. Gagna

dna microarrays
DNA Microarrays

What are they?

  • Microscopic DNA spots representing single genes
  • Allows scientists to observe the interactions between thousands of genes simultaneously
  • Allows for gene expression studies
dna microarrays cont
DNA Microarrays (cont.)

How is this done?

  • DNA spots placed on a solid surface (e.g., glass slide) by covalent attachment
  • Observe the whole genome on a single chip

Result:

  • DNA microarrays allow scientists the ability to study how specific genes work
chromatin immunoprecipitation chip assay
Chromatin Immunoprecipitation(ChIP) Assay

What is it?

  • Special type of DNA microarray

What does it do?

  • Determines whether DNA-binding proteins including transcription factors bind to a specific region of a gene
what is chip on chip
What is “ChIP on ChIP”?

ChIP DNA Microarray

ChIP on ChIP

chip on chip6
ChIP on ChIP

How does it work?

  • Uses a gentle formaldehyde fixation
  • Fixation causes DNA-protein complexes to be seen cross-linked together by formaldehyde
  • DNA-protein complex is isolated and sheared into fragments
  • Antibodies specific for the DNA-binding proteins in question are added so the DNA-protein complex can be isolated
  • DNA and proteins are released so that it can be isolated
chip on chip cont
ChIP on ChIP (cont.)

Result:

  • DNA sequences that can be identified

They can be amplified using Polymerase chain reaction (PCR) method

the problem
The Problem
  • Most scientists have stored their tissue samples in 10% neutral buffered formalin

have overfixed tissue using aldehyde fixatives

purpose of this project
Purpose of this Project
  • To be able to allow scientists to retrieve DNA from overfixed tissue
procedure done
Procedure Done

Materials

  • Model 40 GC lab Oven
  • Crude Extract
  • 96 Microwell
  • Formaldehyde
  • Eosin Stain
  • Hemotoxylin Stain
  • Washing Reagent
  • Microplate Reader
  • Control
procedure done cont
Procedure Done (cont.)

How is done?

  • First 3 columns of microwell were filled with the control
  • In the 5th column of microwell put 40µL of high concentration of extract
  • In the 7th column of microwell put 40µL of low concentration of extract
  • In the 9th column of microwell put 40µL of washing reagent
procedure done cont12
Procedure Done (cont.)
  • With the oven at 50°C, incubated microwell for 20 minutes
  • Stored in the refrigerator at -4°C
  • Staining Chemicals:
    • Hemotoxylin: stains DNA
    • Eosin: stains protein
results before staining
Results: Before Staining
  • Columns 1, 3, 5-control buffer
    • Negative control
  • Column 2-High concentration
    • DNA-protein complex seen
    • In vivo process: cross-linking by formaldehyde
  • Column 4-Low concentration
    • Specks of binding seen
results after staining
Results: After Staining
  • Columns 1, 3, 5-control buffer
    • Negative: no binding
  • Column 2 (top)-eosin stain bound with the protein
  • Column 2 (middle)-controls
    • Little bit of cross containation
  • Column 2 (bottom)- hemotoxylin stain bound with the DNA
  • Column 4 (top)- dark staining by eosin
  • Column 4 (middle)- control
  • Column 4 (bottom)- light staining by hemotoxylin
conclusion
Conclusion
  • Why did column 2 stain darker than column 4?
    • Column 2 had a higher concentration of the extract while column 4 had a low concentration of the extract
  • Why did eosin give a darker staining than the hemotoxylin in column 4?
    • Eosin stains protein
    • When DNA-protein complex made: DNA is surrounded by protein

Therefore, eosin staining will show more the hemotoxylin staining

overall conclusion
Overall Conclusion
  • Our results show that overfixed DNA isolated from tissues can still be successfully retrieved
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